Isothermal assay targeting the V1-V2 region of the 16S rRNA gene for rapid screening of Listeria spp. in raw milk.

Listeria monocytogenes is a foodborne pathogen that, along with L. innocua, serves as a hygiene indicator in food. Different protocols for detecting L. monocytogenes using loop-mediated isothermal amplification (LAMP) have been reported, targeting virulence-associated genes or virulent genes. In contrast, we present a LAMP protocol that targets the V1-V2 regions of the 16S rRNA genes to identify Listeria species in raw milk. The LAMP protocol was standardized using as a template a synthetic version of the V1-V3 region of the 16S rRNA gene from L. monocytogenes. Five protocols for extracting bacterial nucleic acids from raw milk were tested, and boiling in PBS with Tween 20 proved compatible with LAMP. The LAMP assays were tested against fourteen different bacterial monocultures, showing specificity for L. monocytogenes and L. innocua. The RT-LAMP or LAMP method detected up to 100 ag in a 25 μL reaction volume (i.e., 4 ag/μL) of pure 16S rRNA V1-V2 and 16S rDNA V1-V2 from L. monocytogenes, respectively. The LoDs for L. monocytogenes and L. innocua, respectively, were of 10 CFU/mL and 1000 CFU/mL in monocultures or raw milk artificially inoculated. Listeria spp. was detected in 42 % of the raw milk samples tested, and PCR confirmed that 30 % corresponded to L. monocytogenes. The Cohen's Kappa test statistic showed a Kappa value 0.74 (95 % CI: 0.55-0.93) between LAMP and PCR, indicating substantial agreement between both assays. The LAMP protocol shown here does not target a virulence gene; it is specific for detecting Listeria spp. in raw milk, which may carry potential pathogenic risks.